Methane- and methanol-oxidizing bacteria (methylotrophs) are capable of growth on methane or methanol as their sole source of carbon and energy. Their unique metabolic pathways and enzymes have made them a subject of great interest from both biotechnological and environmental perspectives. From a cell biology perspective, their ability to funnel all of their cell carbon through a toxic intermediate (formaldehyde) make them of interest in terms of how they balance formaldehyde production and consumption under a wide regime of substrate flow conditions. It has become clear that one of the mechanisms used is to partition key formaldehyde producing systems into the periplasm. This has resulted in the development of elaborate systems for synthesis, assembly and maturation of complex periplasmic dehydrogenases, which has become a fruitful area of study. However, many questions still remain concerning temporal and spatial aspects of this process, and how all facets are coordinated. This laboratory is particularly interested in gene organization and expression in methylotrophs, and the long-term goal of this project is to understand methylotrophic physiology and how the complex regulons involved in methylotrophy are coordinated in response to the needs of the cell. Under previous NIH support a combined genetic and physiological approach has been used to study C1 metabolism in the facultative methanol-utilizer Methylobacterium extorquens AM1. As a result, we have gained major new insights into four parts of methylotrophic metabolism, methanol oxidation, methylamine utilization, assimilation via the serine cycle and oxidation of formaldehyde. It is now proposed to continue this effort by two approaches. First, our understanding of the genetics of the serine cycle will be extending using a sequencing and metagenesis approach. Second, the regulation of the serine cycle genes, the formaldehyde oxidation genes, and the Mau (methylamine utilization) system will be investigated using transcriptional fusions and where possible, data from these fusions will be compared to data concerning transcripts, transcriptional start sites, protein levels of encoded gene products, and activities of encoded enzymes. Gene fusions will also be used to isolate regulatory genes for the Mau system, which will then be characterized. Similar work has already been carried out for the Mox system, but we propose in addition to search specifically for genes encoding possible negative regulators of the Mox system, and for genes that might coordinate between the different pathways.